Project Summary The hair follicle is a multi-lineage structure that continually undergoes cycles of regression and stem cell- mediated regeneration. Recently, highly-plastic multipotent stem cells have been isolated from both the follicular epithelium and associated dermal stroma. However, the in vivo identity of these cells has not been fully elucidated. The Joyner lab pioneered the technique of genetic inducible fate mapping (GIFM). This technique uses transgenic and gene-targeted mice to express inducible CreER recombinase in specific tissues. Tamoxifen is then administered at a designated time point to induce CreER and permanently activate a ubiquitous reporter allele such as Rosa26R in the CreER-expressing cells. These cells and their daughter cells will subsequently express LacZ. Using GIFM the Joyner lab has demonstrated a role for Sonic Hedgehog (Shh) signaling in the maintenance and expansion of both neural stem cells in the brain and stromal stem cells in the prostate. Based on preliminary observations, Shh regulates both epithelial and stromal stem cells in the cycling hair follicle. Here we propose to investigate the role of Shh in regulating stem cell contributions to the cycling follicle. We will use GIFM of Shh-responding and Shh-expressing cells to test for stem cell traits in these populations. We will also use lineage-specific loss of function studies to block Shh signaling in either the follicular epithelium or the dermis to gauge the relative requirement for Shh signaling in these tissues. Furthermore, we plan to use GIFM to track multiple lineage compartments in the skin to identify the origins of the multipotent skin stem cells derived by culture assays. Genetic labeling and conditional genetic mutation of cells in the skin will allow us to better characterize skin stem cells and the role of molecules, like Shh, that regulate them. This work will also bring us closer to being able to use skin stem cells in treating human disease, and defining their role in cancer and other conditions. Relevance The skin holds great promise as an easily accessible source of adult stem cells for use in the treatment of heritable, destructive and malignant conditions. To better characterize skin stem cells and the molecules that regulate them, we will genetically modify stem cells and follow their growth and maturation. This work will bring us closer to being able to use skin stem cells to treat disease/and defining their role in health, aging and ailments such as cancer.